KR20120036217A - A method of preparing nanofiber for bone tissue regeneration using biopolymer containing gelatin-apatite - Google Patents

Abstract

PURPOSE: A method for fabricating a nanofiber for bone regeneration using biopolymers containing gelatin-apatite is provided to ensure excellent mechanical property and biocompatibility. CONSTITUTION: A method for fabricating a nanofiber for bone regeneration using biopolymers containing gelatin-apatite comprises: a step of preparing gelatin-apatite sol in which apatite precipitate is dispersed in gelatin; a step of freeze-drying the gelatin-apatite sol; a step of mixing the gelatin-apatite with the biopolymers in organic solvent to prepare gelatin-apatite and biopolymers; and a step of electrospinning. The biopolymers include polylactide, coliglycolide, polycaprolactone, polylactide-glycolide copolymers, poly lactide-caprolactone copolymers, poly glycolide-caprolactone copolymer or mixture thereof.

Description

A method of preparing nanofiber for bone tissue regeneration using biopolymer containing gelatin-apatite}

The present invention relates to a method for producing nanofibers for bone tissue regeneration using a biopolymer containing gelatin-apatite, and more particularly, gelatin-apatite- by electrospinning a mixture obtained by mixing gelatin-apatite with synthetic biopolymers. The present invention relates to a method for producing nanofibers for regenerating complex bone tissue composed of biopolymers.

Rebuilding damaged hard tissue using biomedical materials has shown promise in regenerative medicine. Recently, nanofibers have been developed as a new type of scaffold material through electrospinning technology (Liang D et al., Adv. Drug Del. Rev. , 2007, 59, 1392-1412; Pham QP et al., Tiss Eng. , 2006, 12, 1197-1211). Biopolymers including poly (α-hydroxy acids), natural proteins and polysaccharides have been produced as nanofibrous structures with sizes ranging from tens to hundreds of nanometers (Badami AS et al., Biomaterials , 2006, 27, 596-606; Shin M et al., Tiss. Eng. , 2004, 10, 33-41; Yu HS et al., J. Biomed. Mater. Res. A, 2008, 85A, 651-663). The form of the fiber produced has been considered too difficult to obtain by other conventional processing techniques. Furthermore, many biological tests have identified the advantages of nanofibrous structures in cell adhesion and growth and subsequent tissue development (Woo KM et al., Biomater. , 2007, 28, 335-343; Stevens et al., Science , 2005, 310, 1135-1138).

For the regeneration of hard tissues including bones and teeth, recent studies have focused on complexes incorporating inorganic components and polymer matrices. Bone matrices are a type of nanocomposite consisting of apatite nanocrystals and collagen fibrous proteins. Therefore, the complex approach is believed to mimic the natural bone structure. Studies have shown that nanocomposite biomaterials induce better in vitro bone cell responses and in vivo bone formation than single polymers (Kim HW et al., Biomaterials , 2005, 26, 5221-5230; Song JH et al., J. Biomed. Mater. Res. B, 2007, 83B, 248-257; Erisken C et al., Biomater. , 2008, 29, 4065-4073; Kim HW et al., J. Biomed.Mate Res. A, 2008, 85A, 651-663; Lee HH et al., Acta.Biomater . , 2008, 4, 622-629). In particular, porous scaffolds made from hydroxyapatite-precipitated gelatin have been found to significantly enhance bone cell responses. It has also been found that bioactive free components in complexes containing degradable polymers stimulate gene expression and differentiation of bone formation / stem cells. Furthermore, synthetic degradable polymer films incorporating calcium phosphate inorganic phases showed better resistance to rapid degradation associated with acidic environments. However, only limited studies have been carried out to produce nanofibrous matrices made of composites using electrospinning processes. This is because it is too difficult to dry the nanofibrous network from the composite composition compared to a single polymer.

Recently, nanofibrous membranes composed of apatite and gelatin have been produced by electrospinning. Apatite nanocrystals were found to be uniformly distributed in the gelatin matrix when their precipitated products were dissolved in organic solvents. In fact, the concept provides important insights into the fabrication of composite nanofiber systems. However, the rigid and premature dissolution properties of the product must be overcome to produce a matrix that works well as a hard tissue scaffold.

In view of the above, the present inventors prepared a functional composite nanofibrous membrane composed of gelatin-apatite-biopolymer by electrospinning a mixture obtained by mixing gelatin-apatite with a synthetic biopolymer. The present invention has been completed by confirming the excellent mechanical and biological properties.

It is an object of the present invention to provide a method for producing a composite nanofiber composed of gelatin-apatite-biopolymer by electrospinning a mixture obtained by mixing gelatin-apatite with a synthetic biopolymer.

It is another object of the present invention to provide a composite nanofiber composed of gelatin-apatite-biopolymer prepared by the above method, which has improved mechanical properties and biocompatibility.

In one embodiment, the present invention provides a method for preparing a gelatin-apatite sol in which apatite precipitate is dispersed in gelatin; Lyophilizing the gelatin-apatite sol; Mixing the lyophilized gelatin-apatite with biopolymer in an organic solvent to obtain a mixture of gelatin-apatite and biopolymer; And it provides a method for producing a nanofiber for bone tissue regeneration comprising the step of electrospinning the mixture.

The term "apatite" used in the present invention is a kind of phosphate mineral whose chemical formula is Ca ₅ (PO ₄ ) ₃ (OH, F, Cl), also called apatite.

As used herein, the term "biopolymer" means a synthetic polymer having biocompatibility. Specifically, the biopolymers that can be used in the present invention include polylactide, coglycolide, polycaprolactone, or polylactic-glycolide copolymers, polylactide-caprolactone copolymers, and polyglycolide thereof. -Caprolactone copolymers or combinations thereof can be used.

In the present invention, the preparing of the gelatin-apatite sol may be implemented by precipitation reaction of Ca (NO ₃ ) _{2 to} 4H ₂ O and (NH ₃ ) ₂ HPO ₄ in gelatin.

In the present invention, further comprising the step of lyophilizing the gelatin-apatite sol and obtaining a mixture of the gelatin-apatite and biopolymer, washing and lyophilizing the lyophilized gelatin-apatite Can be.

In the present invention, the organic solvent may be trifluoro-ethanol (TFE), hexafluoro-ethanol or a combination thereof.

In the present invention, the mixing ratio of the gelatin-apatite: biopolymer is preferably 1: 4 to 1:10 by weight, most preferably 1: 7. If the mixing ratio is outside the above range, the mechanical properties may be degraded. In particular, when the ratio of gelatin-apatite is higher than the above range, that is, higher than 25% by weight, beads may be formed due to the agglomeration of apatite on the nanofibers, so that mechanical properties may be deteriorated.

In the present invention, the electrospinning method can be carried out through a known method.

In one embodiment of the present invention, a functional nanofiber material composed of gelatin-apatite-poly (lactic-co-caprolactone) (PLCL) was prepared using an electrospinning process. First, a gelatin-apatite precipitate, which mimics the bone extracellular matrix, was prepared, and the precipitate was homogenized with PLCL at various concentrations in organic solvent trifluoro-ethanol (TFE) to obtain a mixture. The membrane was then successfully fabricated by electrospinning the mixture with a fibrous structure having a diameter of several hundred nanometers.

In the experimental example of the present invention, the surface morphology of the membrane prepared as described above was analyzed by scanning electron microscope and transmission electron microscope to confirm that the apatite nanocrystals were effectively distributed in the polymer matrix of gelatin-PLCL. In this case, when a low concentration of gelatin-apatite (14.3% by weight) was mixed, a non-bead-free non-woven nanofibrous web was produced, whereas when a high concentration of gelatin-apatite (25% by weight) was mixed, a significant amount of nanofibers were formed. Beads were observed.

In addition, it was found that by adding a small amount of gelatin-apatite to PLCL, the tensile strength of the nanofibers can be significantly improved up to ˜2 times without losing flexibility.

Furthermore, it was also confirmed that tissue cell growth on the composite nanofibers was greatly improved. Osteoblastic differentiation of cells was significantly stimulated by the composite nanofibers compared to pure PLCL nanofibers.

As a result, when combining the above results, it was confirmed that the gelatin-apatite-PLCL composite nanofiber of the present invention is useful as a bone tissue regeneration matrix.

The present invention has the effect of producing a functional composite nanofiber composed of gelatin-apatite-biopolymer having excellent mechanical properties and biocompatibility by electrospinning the mixture obtained by mixing gelatin-apatite with synthetic biopolymers. Due to the excellent mechanical properties and biocompatibility as described above, the nanofibers of the present invention can be usefully used as a membrane for bone tissue regeneration.

1 is a flow chart briefly illustrating a method for producing a functional composite nanofibrous membrane composed of gelatin-apatite-PLCL of the present invention by electrospinning.
Figure 2 is a result of examining the form of the electrospun nanofiber sheet of the present invention. Where a to c are SEM results, a is pure PLCL, b is PLCL containing low concentration (1/7) of gelatin-apatite precipitate, c is PLCL containing high concentration (1/4) of gelatin-apatite precipitate The form of the nanofiber sheet | seat using this is shown. On the other hand, d is a result of TEM analysis of the appearance of precipitated apatite nanocrystals dispersed in the polymer matrix in the nanofibrous sheet using PLCL containing a low concentration (1/7) gelatin-apatite precipitate.
3 is a result of comparing the mechanical properties by examining the tensile strength of the nanofiber-like composite membrane and pure PLCL membrane. Where a is a representative stress-strain curve of each membrane, b is a graph comparing the maximum tensile stress, and c is the elongation at break (mean ± standard deviation over n = 5) measured with five individual samples. It is a graph shown in comparison. The values obtained from the composite nanofibers were significantly different from those of pure PLCL ( ^* p <0.05 and ^** p <0.01, Student's t -test).
4 shows the response of osteogenic cells to the composite nanofibrous membrane. Where a represents the cell growth morphology on the complex containing low concentration of gelatin-apatite on days 3 and 7, b represents the extent of cell proliferation for up to 7 days as measured by MTS assay, and c is 7 days And at day 14 differentiation of ALP osteogenic cells on nanofibers. Significant differences were observed on the composite nanofibers relative to PLCL ( ^* p <0.01, Student's t -test, n = 3). Significant increases in ALP were observed for incubation time on complexes containing low concentrations of gelatin-apatite (p + <0.01, 7 days versus 14 days).

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples, but these examples are merely illustrative of the present invention, and the scope of the present invention is not limited only to these examples.

Example 1 Preparation of Functional Composite Membranes Made from Gelatin-Apatite-PLCL

A gelatin-apatite sol was prepared by performing a precipitation reaction of Ca (NO ₃ ) _{2 to} 4H ₂ O and (NH ₃ ) ₂ HPO ₄ in gelatin (Type B, bovine skin) according to a previously reported method (Kim HW et al., Adv. Fun.Mater. , 2005, 15, 1988-1989). Briefly stated, two separate Ca-gelatin and P-gelatin solutions were stirred vigorously at 40 ° C. while maintaining a pH of 10 using NH ₄ OH at a ratio of [Ca] / [P] = 1.67. Mixed. The apatite-to-gelatin ratio was maintained at equivalent weight in view of the complete reaction of Ca and P to form apatite. The apatite-precipitated gelatin sol was frozen to −20 ° C. and then lyophilized under vacuum. The dried sample was then washed thoroughly with distilled water / ethanol to remove any salt by-products and then lyophilized. The lyophilized sample was then vigorously stirred for 24 hours to dissolve at 15 w / v% in trifluoro-ethanol (TFE, Aldrich). Poly (lactide-co-caprolactone) (PLCL, Boelinger Ingelheim) dissolved in TFE at 15% by weight was then mixed with the gelatin-apatite solution and two different ratios (gelatin-apatite: PLCL = 1: 4 ( High ratio) and 1: 7 (low ratio)). Each solution was then placed in a syringe and injected onto a rotating mandrel under a high DC voltage of 10 kV at a distance of 15 cm at an injection rate of 0.4 ml / h.

FIG. 1 is a simplified step-by-step process for preparing a functional composite membrane made of the gelatin-apatite-PLCL. Although synthetic polymeric nanofibers, such as PLCL, are good candidates for tissue regeneration, the addition of gelatin-apatite compositions can improve biocompatibility associated with bone tissue. In addition, the apatite inorganic phase can stimulate osteogenic differentiation and calcification when combined with a biopolymer. Moreover, calcium phosphate minerals are known to be very effective in reducing problems associated with acidic products formed during degradation of polymers such as pH reduction and inflammation in vivo. In addition, since the main weakness of synthetic polymers is hydrophobicity and poor cell affinity, the addition of gelatin component can improve the properties of synthetic polymers such as PLCL.

In order to introduce gelatin and apatite components on PLCL, the present invention first synthesized gelatin-apatite precipitates and then homogenized them with PLCL in co-solvent TFE. Compared to the method of adding the individual components (gelatin and apatite) directly, the approach presented in the present invention provided a complex solution with improved mixing properties. It has already been confirmed that a bunch of amino acid groups in gelatin promote spatial homogeneous nucleation of apatite, resulting in more uniform and finer nanocrystals (Kim HW et al., Adv . Fun.Mater. , 2005, 15, 1988-1989). As a result, the electrospun nanofibers prepared from the precipitates had a better fibrous morphology than those produced by direct mixing. In fact, during the electrospinning of the inorganic-organic composite, it is important to use a solution with suitable mixing properties to ensure the production of uniform, bead-free nanofibers.

The addition of gelatin-apatite precipitates to PLCL in this example was carried out at two different concentrations to PLCL, low concentration (1/7 ratio = 14.3 wt%) and high concentration (1/4 ratio = 25 wt%). . The resulting concentrations of apatite were ˜7.2 and 12.5 wt% relative to the PLCL-gelatin polymer phase.

Experimental Example 1: Investigation of the shape of the membrane

The form of the electrospun nanofibers prepared in Example 1 was irradiated with a scanning electron microscope (SEM), and the fiber diameter was measured from the image. Transmission electron microscopy (TEM) was used to determine the presence and distribution of apatite nanocrystals in the nanofibers.

The results are shown in FIG. At both concentrations of gelatin-apatite, spinning into fibrous jets could be performed under controlled conditions. When low concentrations of gelatin-apatite were used (14.3 wt.%, FIG. 2A), a well-developed nonwoven fibrous web with fibers of several hundred nanometers in size was produced. In contrast, when high concentrations of gelatin-apatite were used (25 wt.%, FIG. 2B), some beads were observed and the fiber size was relatively nonuniform. Compared to pure PLCL nanofibers (FIG. 2C), the composite nanofibers had smaller diameters. Specifically, the average fiber size was ~ 780 nm in PLCL, whereas it was ~ 315 nm in high concentrations of gelatin-apatite and -330 nm in low concentrations of gelatin-apatite. The presence of an apatite inorganic phase was evident on the TEM image (FIG. 2D). In addition, highly elongated apatite nanocrystals were well distributed in the PLCL-gelatin matrix and showed no signs of phase separation between the gelatin and PLCL. In view of this, the addition of gelatin-apatite to PLCL was found to be an effective way to obtain nanofibers with well-homogenized inorganic-organic components in the biopolymer matrix.

Experimental Example 2: Investigation of Mechanical Properties of Membrane

Tensile mechanical properties of the nanofibrous membrane prepared in Example 1 were measured using Instron 3344. Membranes were prepared to a thickness of ˜150-200 μm and then cut to a size of 30 mm × 4 mm (gauge length 10 mm) and then subjected to a tensile load. Stress-strain curves were recorded and maximum tensile stress and elongation at break were measured. The thickness of each membrane was determined from the average value observed on the SEM image and a total of five samples were tested for each group.

3 shows the mechanical properties of the composite nanofibers of the present invention compared to pure PLCL nanofibers. Characteristic curves of nanofiber membranes of each composition are shown in FIG. 3A. All nanofibrous membranes showed an initial rapid increase in stress, then the slope decreased and then fractured as the maximum stress value was approached. Maximum stress values (tensile strength) and elongation (%) (strain at break) were obtained from the stress-strain curves shown in FIGS. 3b and 3c. The composite membrane with low concentration of gelatin-PLCL showed the highest strength (average 10.1 MPa), which was almost twice the value of PLCL nanofibers (average 5.7 MPa). However, the strength of the composite with high concentration of gelatin-apatite was slightly lower than the value of pure PLCL (average 4.3 MPa). Although the strain at break of pure PLCL nanofibers was as high as ~ 330%. Due to the addition of gelatin-apatite, this value decreased in a concentration dependent manner, resulting in elongation values of -230% and -90%, respectively, in nanofibers containing low and high concentrations of gelatin-apatite. In gelatin-apatite nanofiber systems, gelatin composite fibers containing 20% and 40% apatite have already been reported to have tensile strengths and elongations of approximately 4-5 MPa and 4-7%, respectively (Kim HW et al. , Adv.Fun. Mater. , 2005, 15, 1988-1989). However, these values were significantly lower than those obtained in the gelatin-apatite-PLCL composite nanofibers of the present invention. Accordingly, it can be seen that the gelatin-apatite-PLCL composite nanofibers of the present invention are significantly superior in mechanical properties compared to the conventional gelatin-apatite nanofibers.

In fact, the addition of inorganic particles onto the polymer is known to enhance mechanical properties when the inorganic particles are finely and uniformly dispersed. In biological systems such as bone, apatite nanocrystals embedded in collagen fibers strengthen and harden bone tissue. In the present invention, it was confirmed that the addition of a small amount of apatite-gelatin is very effective in enhancing the mechanical strength of PLCL nanofibers. It was shown that the ultrafine apatite nanocrystals were uniformly dispersed in the nanofibers, thereby allowing the polymer to withstand the elongation due to the applied load. However, the addition of high concentrations of apatite-gelatin has been found to reduce the strength of PLCL. This is believed to be due to the agglomeration of the apatite nanocrystals, and it can be seen that there are some large beads on the nanofibers (FIG. 2B). Although it can be seen from the initial slope of the curve that there is some reinforcement effect by the weapons phase, the aggregated beads seemed to cause premature failure rather than resistance to the applied load. In contrast, composite nanofibers containing low concentrations of gelatin-apatite exhibited high elongation almost equal to that of pure PLCL nanofibers. Apart from the strengthening effect, maintaining this high flexibility will have a positive effect on the utility of the complex for tissue regeneration membranes or scaffolds for cell growth.

Experimental Example 3: Investigation of Hydrophilicity of Membrane

The hydrophilicity of the nanofiber membranes prepared in Example 1 was examined by measuring the water contact angle (Contact angle analyzer Phoenix300, SEO Co., Korea). Data was recorded up to 1 hour and five samples were tested for each group.

As a result, the hydrophilicity of the composite membrane was found to be significantly higher than that of the PLCL membrane. The initial water contact angle of the nanofiber surface of the PLCL was ˜80 ° and ˜70 ° for the two composites. In addition, water droplets appeared to spread completely onto the composite and penetrated into the nanofiber matrix in ˜1 minute (high concentration of gelatin-apatite) and ˜5 minutes (low concentration of gelatin-apatite). However, even when irradiated with pure PLCL nanofibers, such water penetration was not observed even after 1 hour. This high hydrophilicity of the gelatin-apatite-added PLCL nanofibers allows the material surface to contact the fluid quickly and effectively, enhancing the interaction between biological molecules and cells.

Experimental Example 4 Investigation of Biocompatibility of Membrane

In vitro biocompatibility of the composite nanofibers prepared in Example 1 was observed using osteogenic progenitor cells (MC3T3-E1, ATCC).

Briefly, the cells were passaged in medium consisting of α-MEM supplemented with 10% fetal calf serum (FBS), 2 mM L-glutamine, 50 IU / ml penicillin and 50 μg / ml streptomycin. An electrospun nanofiber web (pure PLCL as a control and two other composites) was prepared in a diameter of 10 mm x 10 mm and then placed in a 24-well plate, followed by the cell suspension (density of 3 x 10 ⁴ cells / ml). ) Was plated on each sample. The samples were then incubated at 37 ° C. and cell growth levels were assessed by MTS assay at 3 and 7 days. The cells were then fixed, dehydrated with a sequential series of ethanol, treated with hexamethyldisilazane, coated with gold, and the cell morphology observed by SEM. The osteogenic differentiation of cells was then examined by measuring the production of alkaline phosphatase (ALP). Cells cultured on each nanofiber sample for a period of 7 days and 14 days were measured using an ALP Activity Kit (Sigma) according to previously reported methods (Kim HW et al., Adv. Fun. Mater. , 2005 , 15, 1988-1989). All of the above cell tests were performed using three samples in each group and the groups were compared by Student's t -test. Significance was considered p <0.05 and p <0.01.

Collectively, the biological role of gelatin-apatite in PLCL nanofibers was assessed in vitro cell growth and bone formation development. To this end, MC3T3-E1 mouse-derived osteogenic progenitor cells were cultured on nanofibrous membranes, and then cell proliferation and alkaline phosphatase concentrations produced in cell structures were examined.

As a result, FIG. 4A shows the morphology of cells grown on the complex (low concentration of gelatin-apatite) and PLCL nanofibrous substrate. On the composite nanofibers, the cells were highly viable early (day 3) with good cytoplasmic expansion, and cells reached nearly confluence, forming a thick cell layer on day 7. On pure PLCL, cells appeared less diffuse than complexes on day 3, but showed similar behavior on day 7. Cell growth levels on the nanofibers were also measured by MTS assay (FIG. 4B). On day 3, cell proliferation was significantly higher on the composite membrane than pure PLCL ( ^* p <0.01). No significant difference was observed between the two composite nanofibers. The initial increase in cell proliferation and growth on the complex is due to their enhanced hydrophilicity, which allows for rapid adsorption of proteins and accelerated cell adhesion processes.

It was confirmed that the alkaline phosphatase concentration was significantly increased in the composite nanofibers (FIG. 4C). ALP enzymatic activity produced on the cell constructs was measured during incubation for 7 and 14 days. In particular, ALP activity of cells grown on complex nanofibers was significantly higher than that of cells grown on pure PLCL on day 7 ( ^* p <0.01), but no significant difference was observed between the two complex nanofibers. At this point, the cells have already reached confluence and formed a thick layer. Therefore, they would have been able to perform osteogenic differentiation, usually associated with the saturated proliferative capacity of MC3T3-E1 cells. After prolonged incubation for 14 days, only a slight increase was found in other membranes, while ALP was significantly (almost doubled) activated in complexes containing low concentrations of gelatin-apatite. As a result, ALP concentrations in complexes containing low concentrations of gelatin-apatite were significantly higher (~ 2 fold) than other membranes. It can be seen that apatite dispersed in the PLCL-gelatin matrix of the nanofibers can enhance osteogenic differentiation, especially during incubation for 7 or 14 days. In addition, apatite synthesized in the presence of gelatin network seems to mimic most of the natural bone. Improvements in early biological events can lead to improvements in the overall process, including cell division and differentiation. In vitro biological stimulation by apatite and gelatin in PLCL compositions has been shown to be similar in the two types of composite nanofibers. Although nanofibers containing high concentrations of gelatin-apatite can stimulate biological activity, some of the large beads formed at these concentrations can have side effects on cellular events.

Taken together with the mechanical properties and the experimental results on in vitro cell response, it can be seen that a small amount of gelatin-apatite can provide optimal substrate conditions for bone tissue regeneration.

Claims (7)

Preparing a gelatin-apatite sol in which the apatite precipitate is dispersed in the gelatin; Lyophilizing the gelatin-apatite sol; Mixing the lyophilized gelatin-apatite with biopolymer in an organic solvent to obtain a mixture of gelatin-apatite and biopolymer; And Method for producing a nanofiber for bone tissue regeneration comprising the step of electrospinning the mixture. The method according to claim 1, wherein the biopolymer is polylactide, coglycolide, polycaprolactone, polylactic-glycolide copolymer, polylactide-caprolactone copolymer, poly glycolide-caprolactone copolymer or Combination method for producing nanofibers for bone tissue regeneration. The method of claim 1 wherein the gelatin comprising the steps of: preparing a cross-linked apatite sol for which the bone tissue implemented by performing a precipitation reaction of _{Ca (NO 3) 2 4H 2} O and (NH _{3) 2} HPO ₄ in the gelatin? Method for producing nanofibers. The method of claim 1, further comprising lyophilizing the gelatin-apatite sol and obtaining a mixture of the gelatin-apatite and biopolymer, further comprising washing and lyophilizing the lyophilized gelatin-apatite. Containing, method for producing nanofibers for bone tissue regeneration. The method of claim 1, wherein the organic solvent is trifluoro-ethanol (TFE), hexafluoro-ethanol, or a combination thereof. The method of claim 1, wherein the gelatin-apatite: biopolymer mixing ratio is 1: 4 to 1:10 by weight ratio. The method of claim 6, wherein the gelatin-apatite: biopolymer mixing ratio is 1: 7 by weight.

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